Immunoglobulin purification

ABSTRACT

The current invention reports a method for purifying an immunoglobulin, wherein the method comprises applying an aqueous, buffered solution comprising an immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby the immunoglobulin in monomeric form does not bind to the cation exchange material, and recovering the immunoglobulin in monomeric form from the solution after the contact with the cation exchange material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation of U.S. patentapplication Ser. No. 15/298,155, filed Oct. 19, 2016, which in turn is acontinuation of U.S. patent application Ser. No. 12/599,703, filed Nov.11, 2009, now U.S. Pat. No. 9,493,548, issued Nov. 15, 2016, which inturn claims priority to National Stage Application of PCT/EP2008/004231,filed May 28, 2008, which claims priority from European Application No.EP07010840.2, filed on Jun. 1, 2007. Each of these applications ishereby incorporated by reference herein in its entirety.

The current invention is in the field of purification of polypeptides.It is reported a method for providing an immunoglobulin in monomericform by separating the immunoglobulin in solution from impurities,especially from the immunoglobulin in aggregated form.

BACKGROUND OF THE INVENTION

Proteins and especially immunoglobulins play an important role intoday's medical portfolio. For human application every therapeuticprotein has to meet distinct criteria. To ensure the safety ofbiopharmaceutical agents to humans nucleic acids, viruses, and host cellproteins, which would cause severe harm, have to be removed especially.To meet these regulatory specifications one or more purification stepshave to follow the manufacturing process. Among other things, purity,throughput, and yield play an important role in determining anappropriate purification process.

Different methods are well established and widespread used for proteinpurification, such as affinity chromatography with microbial proteins(e.g. protein A or protein G affinity chromatography), ion exchangechromatography (e.g. cation exchange (sulfopropyl or carboxymethylresins), anion exchange (amino ethyl resins) and mixed-mode ionexchange), thiophilic adsorption (e.g. with beta-mercaptoethanol andother SH ligands), hydrophobic interaction or aromatic adsorptionchromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, orm-aminophenylboronic acid), metal chelate affinity chromatography (e.g.with Ni(II)- and Cu(II)-affinity material), size exclusionchromatography, and electrophoretical methods (such as gelelectrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A., Appl.Biochem. Biotech. 75 (1998) 93-102).

Necina, R., et al. (Biotechnol. Bioeng. 60 (1998) 689-698) reported thecapture of human monoclonal antibodies directly from cell culturesupematants by ion exchange media exhibiting high charge density. In WO89/05157 a method is reported for the purification of productimmunoglobulins by directly subjecting the cell culture medium to acation exchange treatment. A one-step purification of monoclonal IgGantibodies from mouse ascites is described by Danielsson, A., et al., J.Immun. Meth. 115 (1988), 79-88.

Mhatre, R. et al. (J. Chrom. A 707 (1995) 225-231), explored thepurification of antibody Fab fragments by cation exchange chromatographyand pH gradient elution.

WO 94/00561 reports human monoclonal anti-rhesus antibodies and celllines producing the same. A method for purifying a polypeptide by ionexchange chromatography is reported in WO 2004/024866 in which agradient wash is used to resolve a polypeptide of interest from one ormore contaminants. Schwarz, A. et al. (Laborpraxis 21 (1997) 62-66)report the purification of monoclonal antibodies with aCM-HyperD-column.

WO 2004/076485 reports a process for antibody purification by protein Aand ion exchange chromatography. In EP 0 530 447 a process for purifyingIgG monoclonal antibodies by a combination of three chromatographicsteps is reported. The removal of protein A from antibody preparationsis reported in U.S. Pat. No. 4,983,722.

Recombinant monoclonal antibody processes often employ anion-exchangechromatography to bind trace levels of impurities and potentialcontaminants such as DNA, host cell protein, and virus, while allowingthe antibody to flow through (Knudsen, H. L., et al., J. Chrom. A 907(2001) 145-154).

WO 95/16037 reports the purification of anti-EGF-R/anti-CD3 bispecificmonoclonal antibodies from hybrid hybridoma performed by protein Acation exchange chromatography. The separation of antibody monomers fromits multimers by use of ion exchange chromatography is reported in EP 1084 136. U.S. Pat. No. 5,429,746 relates to the application ofhydrophobic interaction chromatography combination chromatography to thepurification of antibody molecule proteins.

An anionic modified microporous membrane for use for the filtration offluids, particular parenteral or biological liquids contaminated withcharged particulates, is reported in U.S. Pat. No. 4,604,208. WO03/040166 reports a membrane and a device designed for the removal oftrace impurities in protein containing streams.

A method for recovering a polypeptide is reported in U.S. Pat. No.6,716,598. In US 2006/0194953 is reported a method for selectivelyremoving leaked protein A from antibody purified by means of protein Aaffinity chromatography.

SUMMARY OF THE INVENTION

The current invention comprises multiple aspects in the field ofimmunoglobulin purification. One aspect is a method for purifying animmunoglobulin comprising the step of applying an aqueous, bufferedsolution comprising an immunoglobulin in monomeric and in aggregatedform to a cation exchange material under conditions whereby theimmunoglobulin in monomeric form does not bind to the cation exchangematerial, and recovering the immunoglobulin in monomeric form from thesolution after the contact with the cation exchange material. In oneembodiment said step is a chromatographic step operated in flow throughmode.

In one embodiment comprises the method according to the invention afurther step, either prior to or after the cation exchange step, ofapplying an aqueous, buffered solution comprising an immunoglobulin inmonomeric form and/or in aggregated form to an anion exchange materialunder conditions whereby the immunoglobulin does not bind to the anionexchange material, and recovering the immunoglobulin from theflow-through of the anion exchange material. In one embodiment said stepis a chromatographic step operated in flow-through mode.

In another embodiment comprises the method according to the inventioneither prior to the cation exchange step or prior to the anion exchangestep the further step of applying an aqueous, buffered solutioncomprising the immunoglobulin in monomeric and in aggregated form to anaffinity column under conditions whereby the immunoglobulin binds to theaffinity column, and recovering the immunoglobulin in monomeric and inaggregated form from the affinity column. In one embodiment said step isa chromatographic step operated in bind-and-elute mode.

In a further embodiment of the method according to the current inventioncomprises the method as the cation exchange step the step of applying anaqueous, buffered solution comprising the immunoglobulin in monomericand in aggregated form to a membrane cation exchange material underconditions whereby the immunoglobulin in monomeric form does not bind tothe membrane cation exchange material, and recovering the immunoglobulinin monomeric form from the flow-through of the membrane cation exchangematerial. In one embodiment said cation exchange material is a membranecation exchange material. In another embodiment the membrane cationexchange material is Mustang™ S, Mustang™ C, Sartobind™ S, or Sartobind™C. In still another embodiment membrane cation exchange material is apolyethersulfone based membrane or a regenerated cellulose basedmembrane modified with sulfonic acid groups or carboxymethyl groups. Inone embodiment the solution has a pH value of from pH 4 to pH 8,preferably of pH 5 to pH 8. In still another embodiment the solution hasa conductivity of from 1 mS/cm to 15 mS/cm, preferably of from 4.0 mS/cmto 10.0 mS/cm.

Another embodiment comprises that recovering said immunoglobulin inmonomeric form from the flow-through is by a method selected fromprecipitation, salting out, ultrafiltration, diafiltration,lyophilization, affinity chromatography, or solvent volume reduction toobtain a concentrated solution. Preferably said recovering is byultrafiltration, lyophilization, or solvent volume reduction.

In another embodiment said immunoglobulin is obtained from theflow-through of the membrane cation exchange material and at least 95%of the immunoglobulin is in monomeric from. In still another embodimentat least 90% of the immunoglobulin in monomeric form does not bind tothe cation exchange material. One embodiment is that the aqueous,buffered solution is a solution comprising phosphoric acid or saltsthereof, citric acid or salts thereof, or histidine or salts thereof. Inanother embodiment the aqueous, buffered solution comprises sodiumchloride or potassium chloride. And in still another embodiment thechromatographic step is a column chromatography or a cassettechromatography.

DETAILED DESCRIPTION OF THE INVENTION

The current invention comprises a method for purifying an immunoglobulincomprising the step of applying an aqueous, buffered solution comprisingan immunoglobulin in monomeric and in aggregated form to a cationexchange material under conditions whereby the immunoglobulin inmonomeric form does not bind to the cation exchange material, andrecovering the immunoglobulin in monomeric form from the solution orsupernatant after the contact with, i.e. after the removal of, thecation exchange material.

The term “ion exchange material” or grammatical equivalents thereof asused within this application denotes an immobile high molecular weightmatrix that carries covalently bound charged substituents. For overallcharge neutrality not covalently bound counter ions are bound to thecharged substituents by ionic interaction. The “ion exchange material”has the ability to exchange its not covalently bound counter ions forsimilarly charged binding partners or ions of the surrounding solution.Depending on the charge of its exchangeable counter ions the “ionexchange material” is referred to as “cation exchange material” or as“anion exchange material”. Depending on the nature of the charged group(substituent) the “ion exchange material” is referred to as, e.g. in thecase of cation exchange materials, sulfonic acid or sulfopropyl resin(S), or carboxymethyl resin (CM). Depending on the chemical nature ofthe charged group/substituent the “ion exchange material” canadditionally be classified as strong or weak ion exchange material,depending on the strength of the covalently bound charged substituent.For example, strong cation exchange materials have a sulfonic acidgroup, preferably a sulfopropyl group, as charged substituent, weakcation exchange materials have a carboxylic acid group, preferably acarboxymethyl group, as charged substituent. Strong anion exchangematerials have a quarternary ammonium group, and weak anion exchangematerials have a diethylaminoethyl group as charged substituent.

The term “membrane” as used within this application denotes both amicroporous or macroporous membrane. The membrane itself is composed ofa polymeric material such as, e.g. polyethylene, polypropylene, ethylenevinyl acetate copolymers, polytetrafluoroethylene, polycarbonate, polyvinyl chloride, polyamides (nylon, e.g. Zetapore™, N66 Posidyne™),polyesters, cellulose acetate, regenerated cellulose, cellulosecomposites, polysulphones, polyethersulfones, polyarylsulphones,polyphenylsulphones, polyacrylonitrile, polyvinylidene fluoride,non-woven and woven fabrics (e.g. Tyvek®), fibrous material, or of aninorganic material such as zeolithe, SiO₂, Al₂O₃, TiO₂, orhydroxyapatite. In one embodiment is the membrane a polyethersulfonemembrane or a regenerated cellulose membrane.

Ion exchange resins are available under different names and from amultitude of companies such as e.g. cation exchange resins Bio-Rex®(e.g. type 70), Chelex® (e.g. type 100), Macro-Prep® (e.g. type CM, HighS, 25 S), AG® (e.g. type 50W, MP) all available from BioRadLaboratories, WCX 2 available from Ciphergen, Dowex® MAC-3 availablefrom Dow chemical company, Mustang C and Mustang S available from PallCorporation, Cellulose CM (e.g. type 23, 52), hyper-D, partisphereavailable from Whatman plc., Amberlite® IRC (e.g. type 76, 747, 748),Amberlite® GT 73, Toyopearl® (e.g. type SP, CM, 650M) all available fromTosoh Bioscience GmbH, CM 1500 and CM 3000 available from BioChrom Labs,SP-Sepharose™, CM-Sepharose™ available from GE Healthcare, Poros resinsavailable from PerSeptive Biosystems, Asahipak ES (e.g. type 502C),CXpak P, IEC CM (e.g. type 825, 2825, 5025, LG), IEC SP (e.g. type 420N,825), IEC QA (e.g. type LG, 825) available from Shoko America Inc., 50 Wcation exchange resin available from Eichrom Technologies Inc., and suchas e.g. anion exchange resins like Dowex®1 available from Dow chemicalcompany, AG® (e.g. type 1,2,4), Bio-Rex® 5, DEAE Bio-Gel 1, Macro-Prep®DEAE all available from BioRad Laboratories, anion exchange resin type 1available from Eichrom Technologies Inc., Source Q, ANX Sepharose 4,DEAE Sepharose (e.g. type CL-6B, FF), Q Sepharose, Capto Q, Capto S allavailable from GE Healthcare, AX-300 available from PerkinElmer,Asahipak ES-502C, AXpak WA (e.g. type 624, G), IEC DEAE all availablefrom Shoko America Inc., Amberlite® IRA-96, Toyopearl® DEAE, TSKgel DEAEall available from Tosoh Bioscience GmbH, Mustang Q available from PallCorporation. In a membrane ion exchange material the binding sites canbe found at the flow-through pore walls and not hidden within diffusionpores allowing the mass transfer via convection than diffusion. Membraneion exchange materials are available under different names from somecompanies such as e.g. Sartorius (membrane cation exchange material:Sartobind™ CM, Sartobind™ 5, membrane anion exchange material:Sartobind™ Q), or Pall Corporation (membrane cation exchange material:Mustang™ S, Mustang™ C, membrane anion exchange material: Mustang™ Q),or Pall BioPharmaceuticals. In one embodiment the membrane cationexchange material is Sartobind™ CM, or Sartobind™ S, or Mustang™ S, orMustang™ C. In another embodiment the membrane cation exchange materialis a polyethersulfone based membrane or a regenerated cellulose basedmembrane modified with sulfonic acid groups or carboxymethyl groups. Instill another embodiment the anion exchange material is a Q-type(quartemy ammonium group-type) membrane anion exchange material orQ-anion exchange column.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 20 amino acid residues are referred to as “peptides.”

A “protein” is a macromolecule comprising one or more polypeptide chainsor a polypeptide chain of more than 100 amino acid residues. A proteinmay also comprise non-peptidic components, such as carbohydrate groups.Carbohydrate groups and other non-peptidic substituents may be added toa protein by the cell in which the protein is produced, and will varywith the type of cell. Proteins are defined herein in terms of theiramino acid backbone structures; substituents such as carbohydrate groupsare generally not specified, but may be present nonetheless.

The term “immunoglobulin” and grammatical equivalents thereof which canbe used interchangeably within this application comprise at least twolight polypeptide chains and two heavy polypeptide chains. Each of theheavy and light polypeptide chains contains a variable region (generallythe amino terminal portion of the polypeptide chains) which contains abinding domain for interaction with an antigen. Each of the heavy andlight polypeptide chains also comprises a constant region (generally thecarboxyl terminal portion of the polypeptide chains) which may mediatethe binding of the antibody to host tissue or factors including variouscells of the immune system, some phagocytic cells and a first component(C1q) of the classical complement system. Typically, the light and heavypolypeptide chains are chains each consisting essentially of a variableregion, i.e. V_(L) or V_(H), and a constant region, i.e. Of C_(L) incase of a light polypeptide chain, or of C_(H)1, hinge, C_(H)2, C_(H)3,and optionally C_(H)4 in case of a heavy polypeptide chain.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. The recognized immunoglobulin genes include the differentconstant region genes as well as the myriad immunoglobulin variableregion genes. Immunoglobulins may exist in a variety of forms,including, for example, Fv, Fab, and F(ab)₂ as well as single chains(e.g. Huston, J. S., et al., Proc. Natl. Acad. Sci. USA 85 (1988)5879-5883; Bird, R. E., et al., Science 242 (1988) 423-426; and, ingeneral, Hood et al., Immunology, Benjamin N.Y., 2nd edition (1984); andHunkapiller, T., and Hood, L., Nature 323 (1986) 15-16). In oneembodiment immunoglobulins according to the invention comprisemonoclonal antibodies and fragments thereof, for example isolated heavyor light chains, or heavy or light chains fused to a further peptide orpolypeptide, and as well fragments thereof.

The term “immunoglobulin in monomeric form” and grammatical equivalentsthereof denotes an immunoglobulin molecule which is not associated witha second immunoglobulin molecule, i.e. the immunoglobulin molecule isneither covalently nor non-covalently bound to a second immunoglobulinmolecule. The term “immunoglobulin in aggregated form” and grammaticalequivalents thereof denotes an immunoglobulin molecule which isassociated, either covalently or non-covalently, with at least oneadditional immunoglobulin molecule. An immunoglobulin in aggregated formis eluted as a single peak from a size exclusion chromatography column.The term “in monomeric form” and grammatical equivalents thereof as usedwithin this application not necessarily denotes that 100% of animmunoglobulin molecule are present in monomeric form. It furthermoredenotes that of an immunoglobulin at least 90% of the immunoglobulin isin monomeric from, at least 95% of the immunoglobulin is in monomericform, at least 98% of the immunoglobulin is in monomeric form, at least99% of the immunoglobulin is in monomeric form, or more than 99% of theimmunoglobulin is in monomeric form. The term “in monomeric and inaggregated form” denotes a mixture of immunoglobulin molecules notassociated with other immunoglobulin molecules and of immunoglobulinmolecules associated with other immunoglobulin molecules. In thismixture neither of the monomeric form nor the aggregated form is presentexclusively.

The term “100%” as used within this application denotes that the amountof components other than a specified component are below the detectionlimit of the referred to analytical method under the specifiedconditions.

The terms “90%”, “95%”, “98%”, “99%” as used within this applicationdenote no exact values but values within the accuracy of the referred toanalytical method under the specified conditions.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5¹ edition, Part A:Fundamentals and Techniques, Heftmann, E. (ed.), Elsevier SciencePublishing Company, New York, (1992); Advanced Chromatographic andElectromigration Methods in Biosciences, Deyl, Z. (ed.), ElsevierScience BV, Amsterdam, The Netherlands, (1998); Chromatography Today,Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, NewYork, (1991); Scopes, Protein Purification: Principles and Practice(1982); Sambrook, J., et al. (ed.), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; or Current Protocols in Molecular Biology, Ausubel,F. M., et al. (eds), John Wiley & Sons, Inc., New York.

For the purification of recombinantly produced immunoglobulins often acombination of different column chromatographical steps is employed.Generally a protein A affinity chromatography is followed by one or twoadditional separation steps. The final purification step is a so called“polishing step” for the removal of trace impurities and contaminantslike aggregated immunoglobulins, residual HCP (host cell protein), DNA(host cell nucleic acid), viruses, or endotoxins. For this polishingstep often an anion exchange material in a flow-through mode is used.

The term “flow-through mode” and grammatical equivalents thereof as usedwithin the current invention denotes an operation mode of a purificationmethod, e.g. a chromatographic method, in which a solution containing asubstance of interest, e.g. an immunoglobulin in monomeric form, to bepurified is brought in contact with a stationary, preferably solid,phase whereby the substance of interest does not bind to that stationaryphase. As a result the substance of interest is obtained either in theflow-through (if the purification method is a chromatographical method)or the supernatant (if the purification method is a batch method).Substances not of interest, e.g. an immunoglobulin in aggregated form,which were also present in the solution, bind to the stationary phaseand are in that way removed from the solution. This does not necessarilydenote that 100% of the substances not of interest are removed from thesolution but essentially 100% of the substances not of interest areremoved, i.e. at least 50% of the substances not of interest are removedfrom the solution, at least 75% of the substances not of interest areremoved the from solution, at least 90% of the substances not ofinterest are removed from the solution, or more than 95% of thesubstances not of interest are removed from the solution.

The term “applying to” and grammatical equivalents thereof as usedwithin this application denotes a partial step of a purification method,in which a solution containing a substance of interest to be purified isbrought in contact with a stationary phase. This denotes that a) thesolution is added to a chromatographic device in which the stationaryphase is located, or b) that a stationary phase is added to thesolution. In case a) the solution containing the substance of interestto be purified passes through the stationary phase allowing for aninteraction between the stationary phase and the substances in solution.Depending on the conditions, such as e.g. pH, conductivity, saltconcentration, temperature, and/or flow rate, some substances of thesolution are bound to the stationary phase and thus are removed from thesolution. Other substances remain in solution. The substances remainingin solution can be found in the flow-through. The “flow-through” denotesthe solution obtained after the passage of the chromatographic device.Preferably the chromatographic device is a column, or a cassette. In oneembodiment the chromatographic step is a column chromatography, i.e. achromatographic step using a solid phase in a column, or a cassettechromatography, i.e. a chromatographic step using a solid phase in acassette. In preferred embodiment the cassette chromatography employs amembrane in a cassette. The substance of interest not bound to thestationary phase can be recovered in one embodiment from the flow-thoughby methods familiar to a person of skill in the art, such as e.g.precipitation, salting out, ultrafiltration, diafiltration,lyophilization, affinity chromatography, or solvent volume reduction toobtain a concentrated solution. In a preferred embodiment is theimmunoglobulin in monomeric form recovered from the flow-through bylyophilization, ultrafiltration, or solvent volume reduction. In case b)the stationary phase is added, e.g. as a powder, to the solutioncontaining the substance of interest to be purified allowing for aninteraction between the stationary phase and the substances in solution.After the interaction the stationary phase in removed, e.g. byfiltration, and the substance of interest not bound to the stationaryphase is obtained in the supernatant.

The term “does not bind to” and grammatical equivalents thereof as usedwithin this application denotes that a substance of interest, e.g. animmunoglobulin, remains in solution when brought in contact with astationary phase, e.g. an ion exchange material. This does notnecessarily denote that 100% of the substance of interest remains insolution but essentially 100% of the substance of interest remains insolution, i.e. at least 50% of the substance of interest remains insolution and does not bind to the stationary phase, at least 65% of thesubstance of interest remains in solution and does not bind to thestationary phase, at least 80% of the substance of interest remains insolution and does not bind to the stationary phase, at least 90% of thesubstance of interest remains in solution and does not bind to thestationary phase, or more than 95% of the substance of interest remainsin solution and does not bind to the stationary phase.

The term “buffered” as used within this application denotes a solutionin which changes of pH due to the addition or release of acidic or basicsubstances is leveled by a buffer substance. A solution comprising abuffer substance is a “buffered solution”. Any buffer substanceresulting in such an effect can be used. In one embodimentpharmaceutically acceptable buffer substances are used, such as e.g.phosphoric acid and salts thereof, citric acid and salts thereof,morpholine, 2-(N-morpholino) ethanesulfonic acid and salts thereof,Histidine and salts thereof, Glycine and salts thereof, or tris(hydroxymethyl) aminomethane (TRIS) and salts thereof. In a preferredembodiment is the buffer substance phosphoric acid and/or salts thereof,citric acid and/or salts thereof, or histidine and/or salts thereof.Optionally the buffered solution may comprise an additional salt, suchas e.g. sodium chloride, sodium sulphate, potassium chloride, potassiumsulfate, sodium citrate, or potassium citrate. In one embodimentcomprises the buffered solution sodium chloride or potassium chloride.

The term “bind-and-elute mode” as used in the current invention denotesan operation mode of a purification method, in which a solutioncontaining a substance of interest to be purified is brought in contactwith a stationary phase, preferably a solid phase, whereby the substanceof interest binds to the stationary phase. As a result the substance ofinterest is retained on the stationary phase whereas substances not ofinterest are removed with the flow-through or the supernatant. Thesubstance of interest is afterwards optionally after a washing stepeluted from the stationary phase in a second step and thereby recoveredfrom the stationary phase with an elution solution.

For the separation of immunoglobulins in monomeric form fromimmunoglobulins in aggregated form generally chromatographic methodsemploying cation exchange materials operated in a bind-and-elute modeare used, see e.g. WO 2006/125599. It has now surprisingly been foundthat cation exchange materials operated in a flow-through mode can beused for the removal of immunoglobulins in aggregated form fromsolutions containing immunoglobulins in monomeric and aggregated form.With this method for purifying an immunoglobulin it is possible toremove immunoglobulins in aggregated form in a fast and easy way fromsolutions containing a mixture of immunoglobulins both in monomeric formand in aggregated form.

Thus, the current invention reports a method for purifying animmunoglobulin, wherein the method comprises the following step:

a) applying an aqueous, buffered solution comprising an immunoglobulinin monomeric and in aggregated form to a cation exchange material underconditions whereby the immunoglobulin in monomeric form does not bind tothe cation exchange material, and recovering the immunoglobulin inmonomeric form from the solution after the contact with the cationexchange material.

In more detail, the current invention comprises a method for obtainingan immunoglobulin in monomeric form from a solution comprising theimmunoglobulin in monomeric and in aggregated form, whereby the methodcomprises the step of applying an aqueous, buffered solution comprisingthe immunoglobulin in monomeric and in aggregated form to a cationexchange material under conditions whereby the immunoglobulin inmonomeric form does not bind to the cation exchange material, andrecovering the immunoglobulin in monomeric form from the solution afterthe contact with the cation exchange material. In one embodiment saidstep is a chromatographic step operated in flow-through mode. In anotherembodiment is said cation exchange material a membrane cation exchangematerial.

The term “conditions under which the immunoglobulin in monomeric formdoes not bind to the cation exchange material” and grammaticalequivalents thereof as used within this application denotes that animmunoglobulin in monomeric form remains in solution when brought incontact with the cation exchange material. This does not necessarilydenote that 100% of the immunoglobulin in monomeric form remains insolution but essentially 100% of the immunoglobulin in monomeric formremains in solution and does not bind to the cation exchange material,i.e. at least 50% of the immunoglobulin in monomeric form remains insolution and does not bind to the cation exchange material, at least 65%of the immunoglobulin in monomeric form remains in solution and does notbind to the cation exchange material, at least 80% of the immunoglobulinin monomeric form remains in solution and does not bind to the cationexchange material, at least 90% of the immunoglobulin in monomeric formremains in solution and does not bind to the cation exchange material,or more than 95% of the immunoglobulin in monomeric form remains insolution and does not bind to the cation exchange material. Suchconditions are e.g. in one embodiment a pH value of the aqueous,buffered solution of from pH 5 to pH 8 and/or in another embodiment aconductivity of the aqueous, buffered solution of from 1.0 mS/cm to 15mS/cm, preferably of from 4.0 mS/cm to 10.0 mS/cm.

In one embodiment of the current invention comprises the method forpurifying an immunoglobulin the step of applying an aqueous, bufferedsolution comprising the immunoglobulin in monomeric and in aggregatedform to a membrane cation exchange material under conditions whereby theimmunoglobulin in monomeric form does not bind to the membrane cationexchange material, and recovering the immunoglobulin in monomeric formfrom the flow-through of the membrane cation exchange material.

In this embodiment the purification method is a chromatographic methodoperated in flow-through mode, which allows for a rapid purification ofthe immunoglobulin, because the desired immunoglobulin in monomeric formcan easily be obtained from the flow-through of the column, makingfurther steps, such as washing of the column, elution of the boundsubstance, or desalting of the eluted immunoglobulin solution,unnecessary.

The method according to the invention can be employed as a single stepmethod or combined with other steps, such as, e.g., in one embodimentwith an anion exchange chromatography step, or with an affinitychromatography step in another embodiment.

Thus, one embodiment of the current invention is a method for purifyingan immunoglobulin, wherein the method comprises the following sequentialsteps:

b) applying an aqueous, buffered solution comprising the immunoglobulinin monomeric and in aggregated form to an anion exchange chromatographycolumn under conditions whereby the immunoglobulin does not bind to theanion exchange material, and recovering the immunoglobulin in monomericand in aggregated form from the flow-through of the anion exchangecolumn as an aqueous, buffered solution, and

a) applying the aqueous, buffered solution obtained in step b)comprising an immunoglobulin in monomeric and in aggregated form to acation exchange material under conditions whereby the immunoglobulin inmonomeric form does not bind to the cation exchange material, andrecovering the immunoglobulin in monomeric form from the solution afterthe contact with the cation exchange material.

In one embodiment step b) is a chromatographic method operated inflow-through mode. In another embodiment step a) is a chromatographicmethod operated in flow-through mode. In another embodiment is saidcation exchange material in step a) a membrane cation exchange material.The conditions for step b) are known to a person of skill in the art.With step b) it is possible to reduce the amount of host cell protein(HCP), protein A, endotoxins, and/or viruses in the solution containingthe immunoglobulin. It is also possible to reverse the order of the twoion exchange steps. Prior to the application of a solution to one step(or to a subsequent step) of a purification method parameters, such ase. g. the pH value or the conductivity of the solution, have to beadjusted. In one embodiment the pH value of the aqueous solution appliedin step a) is of from pH 4 to pH 8, preferably of from pH 5 to pH 8. Inan other embodiment the conductivity of the aqueous solution is of from4.0 mS/cm to 10.0 mS/cm.

Additionally, the current invention comprises in one embodiment a methodfor purifying an immunoglobulin, wherein the method comprises thefollowing sequential steps:

a) applying an aqueous, buffered solution comprising an immunoglobulinin monomeric and in aggregated form to a cation exchange material underconditions whereby the immunoglobulin in monomeric form does not bind tothe cation exchange material, and recovering the immunoglobulin inmonomeric form from the solution after the contact with the cationexchange material, and

c) applying the aqueous, buffered solution of step a) comprising theimmunoglobulin in monomeric form to an anion exchange chromatographycolumn under conditions whereby the immunoglobulin does not bind to theanion exchange material, and recovering the immunoglobulin in monomericform from the flow-through of the anion exchange chromatography column.

In one embodiment step c) is a chromatographic method operated inflow-through mode. In another embodiment step a) is a chromatographicmethod operated in flow-through mode. In another embodiment is saidcation exchange material in step a) a membrane cation exchange material.It might be helpful e.g. to remove the bulk of the host cell proteinsand culture by-products in a foremost purification step employing anaffinity chromatography.

One embodiment of the current invention is a method comprising thefollowing steps in this order:

d) applying an aqueous, buffered solution comprising the immunoglobulinin monomeric and in aggregated form to an affinity column underconditions whereby the immunoglobulin binds to the affinity column, andrecovering the immunoglobulin in monomeric and in aggregated form fromthe affinity column as a aqueous, buffered solution, and

b) optionally applying the aqueous, buffered solution obtained in stepd) comprising the immunoglobulin in monomeric and in aggregated form toan anion exchange chromatography column under conditions whereby theimmunoglobulin does not bind to the anion exchange material, andrecovering the immunoglobulin in monomeric and in aggregated form fromthe flow-through of the anion exchange column as an aqueous, bufferedsolution, and

a) applying the aqueous, buffered solution either obtained in step d) orobtained in step b) comprising an immunoglobulin in monomeric and inaggregated form to a cation exchange material under conditions wherebythe immunoglobulin in monomeric form does not bind to the cationexchange material, and recovering the immunoglobulin in monomeric formfrom the solution after the contact with the cation exchange material.

In one embodiment step b) is a chromatographic method operated inflow-through mode. In another embodiment step a) is a chromatographicmethod operated in flow-through mode. In another embodiment step d) isoperated in bind-and-elute mode. In another embodiment is said cationexchange material a membrane cation exchange material. The affinitycolumn may e.g. be a protein A affinity column, a protein G affinitycolumn, a hydrophobic charge induction chromatography column (HCIC), ora hydrophobic interaction chromatography column (HIC, e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid).Preferably the affinity column is a protein A column or a HCIC column.

In a further embodiment the aqueous buffered solution comprising theimmunoglobulin in monomeric form and in aggregated form in step b)and/or the aqueous buffered solution comprising the immunoglobulin inmonomeric form in step c) is applied to an anion exchange chromatographycolumn under conditions whereby the immunoglobulin does bind to theanion exchange material, and is recovered from the anion exchangematerial as a aqueous, buffered solution.

One aspect of the invention is a method for obtaining an immunoglobulinin monomeric form from a solution comprising the immunoglobulin inmonomeric and in aggregated form, whereby this method comprises thefollowing step:

-   -   a) a chromatographic step operated in flow-through mode        comprising applying an aqueous, buffered solution comprising the        immunoglobulin in monomeric form and in aggregated form to a        membrane cation exchange material under conditions whereby the        immunoglobulin in monomeric form does not bind to the cation        exchange material, the immunoglobulin in monomeric form is        recovered from the flow-through of the membrane cation exchange        material.

In one embodiment the method according to the invention comprises priorto step a) the following step:

-   -   b) a chromatographic step operated in flow-through mode        comprising applying an aqueous, buffered solution comprising the        immunoglobulin in monomeric and in aggregated form to an anion        exchange chromatography column under conditions whereby the        immunoglobulin does not bind to the anion exchange material, and        recovering the immunoglobulin in monomeric and in aggregated        form from the flow-through of the anion exchange column as an        aqueous, buffered solution.

In one embodiment the method according to the invention comprises afterstep a) the following step:

-   -   c) a chromatographic step operated in flow-through mode        comprising applying the aqueous, buffered solution obtained in        step a) comprising the immunoglobulin in monomeric form to an        anion exchange chromatography column under conditions whereby        the immunoglobulin does not bind to the anion exchange material,        and recovering the immunoglobulin in monomeric form from the        flow-through of the anion exchange chromatography column.

In another embodiment the method according to the invention comprisesprior to step a) or step b) the following step:

-   -   d) a chromatographic step operated in bind-end-elute mode        comprising applying an aqueous, buffered solution comprising the        immunoglobulin in monomeric and in aggregated form to an        affinity column under conditions whereby the immunoglobulin        binds to the affinity column, and recovering the immunoglobulin        in monomeric and in aggregated form from the affinity column as        a aqueous, buffered solution.

In another embodiment the recovering said immunoglobulin in monomericform from the flow-through is by a method selected from precipitation,salting out, ultrafiltration, diafiltration, lyophilization, affinitychromatography, or solvent volume reduction to obtain a concentratedsolution. Preferably said recovering is by ultrafiltration,lyophilization, or solvent volume reduction.

In one embodiment the immunoglobulin is obtained from the flow-throughof the membrane cation exchange material at least 95% of theimmunoglobulin is in monomeric from. In another embodiment at least 90%of the immunoglobulin in monomeric form does not bind to the cationexchange material.

In still another embodiment the pH value of the aqueous, bufferedsolution is of from pH 5 to pH 8. In a further embodiment theconductivity of the aqueous, buffered solution of from 4.0 mS/cm to 10.0mS/cm. In one embodiment the buffered solution is a solution comprisingphosphoric acid or salts thereof, citric acid or salts thereof, orhistidine or salts thereof. In still another embodiment the aqueoussolution comprises sodium chloride or potassium chloride.

In one embodiment the membrane cation exchange material is apolyethersulfone based membrane or a regenerated cellulose basedmembrane modified with sulfonic acid groups or carboxymethyl groups. Ina further embodiment the chromatographic step is a column chromatographyor a cassette chromatography.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: Diagram of mAb IL13 flow-through mode purification with aMustang™ S membrane.

FIG. 2A: SEC (size exclusion chromatogram) analysis of the samplematerial mAb IL13.

FIG. 2B: SEC analysis of fraction 2 of the flow-through.

FIG. 3: SEC analysis of fraction 2 of the flow-through of a Sartobind™ Cmembrane process of sample material containing mAb IL13.

FIG. 4: SEC analysis of fraction 4 of the flow-through of a Sartobind™ Cmembrane process of sample material containing of mAb Her2.

EXAMPLES

Materials and Methods:

Conditioned Protein a Eluate:

An anti-(IL-13Rα1) antibody (hereinafter referred to as mAb IL13, seee.g. WO 2006/072564) and an anti-Her2 antibody (hereinafter referred toas mAb Her2, see e.g. U.S. Pat. No. 5,677,171) were purified in a firststep with a protein A affinity chromatography.

The mAb IL13 was eluted from the protein A column under acidicconditions (3.5 mM hydrochloric acid, pH value 2.7±0.2). Before thefiltration step the pH value of the fraction containing theimmunoglobulin was adjusted with a concentrated, e.g. 1 M, buffersolution of pH 9.0 (e.g. tris (hydroxymethyl) aminomethane (TRIS) orphosphate buffer) to pH 5.0. This material is referred to in thefollowing as conditioned protein A eluate of mAb IL13.

The mAb Her2 was purified in a first step with a protein A affinitychromatography. Elution from the protein A column is carried out underacidic conditions (10 mM sodium citrate buffer, pH value of 3.0±0.5).Before the filtration step the pH value of the fraction containing theimmunoglobulin is adjusted with a concentrated tris(hydroxymethyl)aminomethane (TRIS) buffer to pH 5.6. This material isreferred to in the following as conditioned protein A eluate of mAbHer2.

Analytical Methods:

Size Exclusion Chromatography:

resin: TSK 3000 (Tosohaas) column: 300 × 7.8 mm flow rate: 0.5 ml/minbuffer: 200 mM potassium phosphate containing 250 mM potassium chloride,adjusted to pH 7.0 wavelength: 280 nm DNA- see e.g. Merrick, H., andHawlitschek, G., Biotech threshold- Forum Europe 9 (1992) 398-403system: Protein A The wells of a micro titer plate are coated with aELISA: polyclonal anti-protein A-IgG derived from chicken. After bindingnon-reacted antibody is removed by washing with sample buffer. Forprotein A binding a defined sample volume is added to the wells. Theprotein A present in the sample is bound by the chicken antibody andretained in the wells of the plate. After the incubation the samplesolution is removed and the wells are washed. For detection are addedsubsequently a chicken derived polyclonal anti- protein A-IgG-biotinconjugate and a Streptavidin peroxidase conjugate. After a furtherwashing step substrate solution is added resulting in the formation of acolored reaction product. The intensity of the color is proportional tothe protein A content of the sample. After a defined time the reactionis stopped and the absorbance is measured. Host cell The walls of thewells of a micro titer plate are coated protein with a mixture of serumalbumin and Streptavidin. A (HCP) ELISA: goat derived polyclonalantibody against HCP is bound to the walls of the wells of the microtiter plate. After a washing step different wells of the micro titerplate are incubated with a HCP calibration sequence of differentconcentrations and sample solution. After the incubation not boundsample material is removed by washing with buffer solution. For thedetection the wells are incubated with an antibody peroxidase conjugateto detect bound host cell protein. The fixed peroxidase activity isdetected by incubation with ABTS and detection at 405 nm.

Example 1

Purification of mAb IL13 to Monomeric Form—Comparison of Conditions

In the purification of mAb IL13 to monomeric form different conditionswere evaluated.

The purification method was operated as a chromatographic purificationmethod in flow-through mode. Different conditions for the flow-throughpurification of the mAb IL13 were evaluated. As membrane cation exchangematerial the Mustang™ S-Adsorber System (Mustang™ S Coin, 1.5 cm²membrane area, Pall Corporation, USA) has been used.

The conditioned protein A eluate had a mAb IL13 concentration of 7.1 g/lwith 90.9% of the immunoglobulin in monomeric form and 9.1% of theimmunoglobulin in aggregated form. The conditioned protein A eluate hasbeen virus inactivated and filtered through a 0.2 μm pore filter priorto the experiments. After dilution with the corresponding buffer to aprotein concentration of approx. 1 mg/ml (a ratio of approx. 1:7 (v/v)),pH and conductivity adjustment, the solutions were applied to themembrane cation exchange material. If necessary the adjustment of pH wasperformed with potassium hydrogen phosphate or potassiumdihydrogenphosphate and conductivity adjustment was done by the additionof KCl or deionized water (with pH of approx. 5.5 and 7.5,respectively). Such a diluted, adjusted, and conditioned protein Aeluate is referred to in the following as sample material.

Diversified parameters were conductivity and pH. Observed parameterswere yield and purity of the flow-through immunoglobulin in monomericform of the sample material. The diversified parameters of the samplematerial are summarized in Table 1.

TABLE 1 Sample material parameters Sample pH conductivity No. Valuebuffer [mS/cm] 1 5.5 40 mM potassium phosphate 4.8 2 6.5 40 mM potassiumphosphate 4.8 3 7.5 40 mM potassium phosphate 4.8 4 5.5 50 mM potassiumphosphate 5.8 5 6.5 50 mM potassium phosphate 5.8 6 7.5 50 mM potassiumphosphate 5.8 7 5.5 60 mM potassium phosphate 6.8 8 6.5 60 mM potassiumphosphate 6.8 9 7.5 60 mM potassium phosphate 6.8

The flow-through has been analyzed by size exclusion chromatography inorder to determine the amount of immunoglobulin in monomeric form and inaggregated form. The second fraction was chosen for analysis because inthe beginning of the process (1^(st) fraction) no stable purificationprocess had established because due to the dead volume of thechromatographic system no steady flow exists and thus an unknowndilution of the 1^(st) fraction occurs. An exemplary flow-throughdiagram is depicted in FIG. 1. The results are summarized in Table 2.The size exclusion chromatograms of the sample material and of fraction2 (pH 6.5, 5.8 mS/cm) are shown in FIGS. 2A and 2B.

TABLE 2 mAb IL13 immunoglobulin in monomeric form (as area percentage ofSEC chromatography) and yield with a Mustang ™ S membrane. pH value 5.56.5 7.5 conductivity mAb IL13 mAb IL13 mAb IL13 [mS/cm] monomeric formyield monomeric form yield monomeric form yield 4.8 100% 33.6% 100%82.5% 75% 94.4% 5.8 100% 65.7% 100% 90.9%  0%  100% 6.8 100% 67.9%  99%91.1%  0% —

The data presented in Table 2 show that conditions suitable for thepurification of mAb IL13, i.e. the separation of an immunoglobulin inmonomeric form from the aggregated form, i.e. conditions under which theimmunoglobulin in monomeric form does not bind to the membrane cationexchange material, with excellent yield can be adjusted, such as e.g. aconductivity of 5.8 mS/cm and a pH of 6.5.

Example 2

Purification of mAb IL13 to Monomeric Form—Comparison of Membranes

The results obtained in Example 1 are generally applicable and have beenapplied to the membrane cation exchange material Sartobind™ S (75 cm²membrane area, Sartorius AG, Göttingen, Germany). The preferredconditions for Mustang™, i.e. conductivity of 5.8 mS/cm at a pH of 6.5,were also applied to the Sartobind™ material. The sample materialcontaining mAb IL13 had a protein concentration of 1.34 mg/ml with 94.8%of the immunoglobulin in monomeric form and 5.2% of the immunoglobulinin aggregated form. The sample material was applied to the membrane asreported in Example 1. The results of the purification process aresummarized in Table 3.

TABLE 3 mAb IL13 immunoglobulin in monomeric and aggregated form (asarea percentage of SEC chromatography) and yield with a Sartobind ™ Smembrane. Sample mAb IL13 in amount mAb IL13 in monomeric aggregatedform in of form in flow-through flow-through Sample mAb Sample totalconcentration amount amount No. IL13 volume area percentage yield areapercentage 1 14.71 mg 11 ml 0.029 mg/ml  100%  1.02 mg  6.9% bdl — 214.71 mg 11 ml  0.51 mg/ml   99%  5.59 mg 38.0% 1.00% 0.06 mg 3 14.71 mg11 ml  0.89 mg/ml 97.6%  9.54 mg 64.9% 2.45% 0.24 mg 4 14.71 mg 11 ml 0.94 mg/ml 97.5% 10.03 mg 68.2% 2.53% 0.26 mg 5 14.71 mg 11 ml  0.94mg/ml 97.8% 10.11 mg 68.7% 2.17% 0.22 mg 6 14.71 mg 11 ml  0.94 mg/ml97.9% 10.16 mg 69.1% 2.07% 0.22 mg 7 14.71 mg 11 ml  0.95 mg/ml 97.5%10.14 mg 68.9% 2.54% 0.26 mg Average 14.71 mg 11 ml  0.94 mg/ml 97.7%10.11 mg 68.7% 2.33% 0.24 mg of samples 4 to 7 bdl = below detectionlimit The same sample material was also applied to a Sartobind ™ Cmembrane. The results are presented in Table 4 and an exemplary sizeexclusion chromatogram of the 3^(rd) fraction is shown in FIG. 3.

TABLE 4 mAb IL13 immunoglobulin in monomeric and aggregated form (asarea percentage of SEC chromatography) and yield with a Sartobind ™ Cmembrane. Sample mAb IL13 in amount mAb IL13 in monomeric aggregatedform in of form in flow-through flow-through Sample mAb Sample totalconcentration amount amount No. IL13 volume area percentage yield areapercentage 1 14.71 mg 11 ml    0 mg/ml bdl — — bdl — 2 14.71 mg 11 ml0.033 mg/ml  100%  0.36 mg  2.4% bdl — 3 14.71 mg 11 ml  0.56 mg/ml 100%  6.15 mg 41.8% bdl — 4 14.71 mg 11 ml  0.87 mg/ml 99.4%  9.54 mg64.9% 0.65% 0.06 mg 5 14.71 mg 11 ml  0.93 mg/ml 99.3% 10.20 mg 69.3%0.66% 0.07 mg 6 14.71 mg 11 ml  0.96 mg/ml 98.9% 10.43 mg 70.9% 1.13%0.12 mg 7 14.71 mg 11 ml  0.98 mg/ml 98.9% 10.66 mg 72.5% 1.08% 0.12 mgAverage 14.71 mg 11 ml  0.94 mg/ml 99.1% 10.21 mg 69.4% 0.88% 0.09 mg ofsamples 4 to 7 bdl = below detection limit — = not determined By usingthe purification conditions determined with one membrane material in allexperiments, all cationic membrane adsorbers demonstrated will have thecapability to remove aggregates and to obtain monomeric IgG inflow-through mode operation with the same conditions.

Example 3

Purification of mAb Her2 to Monomeric Form—Comparison of Membranes

The conditioned protein A eluate had a mAb Her2 concentration of 7.61g/l with 98.8% purity. The immunoglobulin in aggregated form wasproduced/obtained by heating of the mAb Her2 solution to 37° C. for 3days. The solution contained after the heat treatment 99.0% of theimmunoglobulin in monomeric form and 1.0% of the immunoglobulin inaggregated form without considering low molecular weight substancespresent in the heat treated sample. The conditioned protein A eluate hadbeen virus inactivated and filtered through a 0.2 μm pore size filterprior to the experiments. After dilution to protein concentration of1.03 mg/ml, pH and conductivity adjustment, the solution had beenapplied to the membrane cation exchange materials Sartobind™ S and C,respectively. The adjustment of conductivity was done by the addition ofNaCl (5 mol/l). The results are shown in Tables 5 and 6.

TABLE 5 mAb Her2 immunoglobulin in monomeric and aggregated form (asarea percentage of SEC chromatography) and yield with a Sartobind ™ Smembrane. Sample mAb Her2 in amount mAb Her2 in monomeric aggregatedform in of form in flow-through flow-through Sample mAb Sample totalconcentration amount amount No. Her2 volume area percentage yield areapercentage 1 11.35 mg 11 ml 0.053 mg/ml 40.4%  0.23 mg 0.02% bdl — 211.35 mg 11 ml 0.600 mg/ml 99.2%  6.47 mg 57.0% bdl — 3 11.35 mg 11 ml0.908 mg/ml 99.8%  9.86 mg 86.9% 0.02% 0.002 mg 4 11.35 mg 11 ml 0.977mg/ml 99.9% 10.60 mg 93.4% 0.03% 0.003 mg 5 11.35 mg 11 ml 0.999 mg/ml99.8% 10.83 mg 95.4% bdl — 6 11.35 mg 11 ml 1.013 mg/ml 99.9% 10.99 mg96.8% bdl — 7 11.35 mg 11 ml 1.019 mg/ml 99.8% 10.04 mg 88.5% 0.05%0.006 mg 8 11.35 mg 11 ml 1.023 mg/ml 99.9% 11.10 mg 97.8% 0.08% 0.009mg 9 11.35 mg 11 ml 1.025 mg/ml 99.8% 11.12 mg 98.0% 0.14% 0.016 mg 1011.35 mg 11 ml 1.024 mg/ml 99.8% 11.11 mg 97.9% 0.17% 0.019 mg 11 11.35mg 11 ml 1.025 mg/ml 99.7% 11.11 mg 97.9% 0.18% 0.020 mg 12 11.35 mg 11ml 1.027 mg/ml 99.7% 11.13 mg 98.1% 0.23% 0.026 mg 13 11.35 mg 11 ml1.028 mg/ml 99.7% 11.13 mg 98.1% 0.24% 0.027 mg 14 11.35 mg 11 ml 1.028mg/ml 99.6% 11.13 mg 98.1% 0.30% 0.034 mg 15 11.35 mg 11 ml 1.027 mg/ml99.6% 11.30 mg 99.6% 0.32% 0.036 mg Average 11.35 mg 11 ml 1.018 mg/ml99.8% 10.97 mg 96.7% 0.14% 0.016 mg of samples 4 to 15 bdl = belowdetection limit — = not determined

TABLE 6 mAb Her2 immunoglobulin in monomeric and aggregated form (asarea percentage of SEC chromatography) and yield with a Sartobind ™ Cmembrane. Sample mAb Her2 in amount mAb Her2 in monomeric aggregatedform in of form in flow-through flow-through Sample mAb Sample totalconcentration amount amount No. Her2 volume area percentage yield areapercentage 1 11.35 mg 11 ml 0.082 mg/ml 74.8%  0.67 mg  5.9% bdl — 211.35 mg 11 ml 0.656 mg/ml 99.1%  7.07 mg 62.3% bdl — 3 11.35 mg 11 ml0.861 mg/ml 99.7%  9.32 mg 82.1% bdl — 4 11.35 mg 11 ml 0.929 mg/ml99.7% 10.06 mg 88.6% 0.04% 0.004 mg 5 11.35 mg 11 ml 0.965 mg/ml 99.7%10.45 mg 92.1% 0.09%  0.01 mg 6 11.35 mg 11 ml 0.992 mg/ml 99.7% 10.47mg 92.2% 0.15% 0.015 mg 7 11.35 mg 11 ml 1.004 mg/ml 99.8% 10.88 mg95.9% 0.14% 0.015 mg 8 11.35 mg 11 ml 1.014 mg/ml 99.7% 10.98 mg 96.7%0.24% 0.027 mg 9 11.35 mg 11 ml 1.015 mg/ml 99.7% 10.99 mg 96.8% 0.29%0.032 mg 10 11.35 mg 11 ml 1.017 mg/ml 99.6% 11.00 mg 96.9% 0.36%  0.04mg 11 11.35 mg 11 ml 1.019 mg/ml 99.6% 11.03 mg 97.2% 0.43% 0.047 mg 1211.35 mg 11 ml 1.021 mg/ml 99.5% 11.04 mg 97.3% 0.41% 0.046 mg 13 11.35mg 11 ml 1.022 mg/ml 99.5% 11.05 mg 97.4% 0.42% 0.047 mg 14 11.35 mg 11ml 1.024 mg/ml 99.5% 11.07 mg 97.5% 0.51% 0.055 mg 15 11.35 mg 11 ml1.024 mg/ml 99.5% 11.07 mg 97.5% 0.47% 0.051 mg Average 11.35 mg 11 ml 1.0 mg/ml 99.6% 10.84 mg 95.5% 0.30% 0.032 mg of samples 4 to 15 bdl =below detection limit — = not determined An exemplary size exclusionchromatography (SEC) analysis of fraction 4 of the flow-through of aSartobind C membrane is shown in FIG. 4.

It can be summarized from the above that the sum of pH value andconductivity in mS/cm is preferably in the range from 9 to 18, morepreferably in the range from 10 to 15.

Example 4

Analysis of Protein a, DNA and HCP Content.

The fractions obtained in Example 2 have been analyzed for protein A-,DNA-, and HCP-content. The results are given in Tables 7 and 8.

The protein A-content in the solution prior to the application to theSartobind™ S membrane was 2.6 ng/ml, the DNA-content was <0.3 pg/mg, andthe host cell protein (HCP)-content was 1791 ng/mg.

TABLE 7 Protein A-, DNA-, and HCP-content of fractions containing mAbIL13 immunoglobulin obtained with a Sartobind ™ S membrane. SampleProtein Sample volume concentration protein A DNA HCP No. [ml] [mg/ml][ng/mg] [pg/mg] [ng/mg] 1 11 0.029 <4.1 <138 28 2 11 0.513 0.5 <0.8 <1183 11 0.889 <0.3 <0.4 <202 4 11 0.935 0.7 <0.4 255 5 11 0.939 — <0.4 — 611 0.943 — <0.4 — — = not determined The protein A-content in thesolution prior to the application to the Sartobind ™ C membrane was 3.0ng/ml, the DNA-content was <0.4 pg/mg, and the host cell protein(HCP)-content was 5250 ng/mg.

TABLE 8 Protein A-, DNA-, and HCP-content of fractions containing mAbIL13 immunoglobulin obtained with a Sartobind ™ C membrane. SampleProtein Sample volume concentration protein A DNA HCP No. [ml] [mg/ml][ng/mg] [pg/mg] [ng/mg] 1 11 0.033 <4.2 <13.3 155 2 11 0.559 <0.2 <0.71036 3 11 0.873 0.3 <0.5 989 4 11 0.933 0.7 <0.4 1366 5 11 0.959 n.b.n.b. n.b. 6 11 0.979 n.b. n.b. n.b.

The invention claimed is:
 1. A method for obtaining an immunoglobulin inmonomeric form from a solution comprising the immunoglobulin inmonomeric and in aggregated form, comprising the steps of: 1) applyingan aqueous, buffered solution comprising said immunoglobulin inmonomeric and in aggregated form to an affinity column under conditionswhereby said immunoglobulin binds to said affinity column, andrecovering said immunoglobulin in monomeric and in aggregated form fromsaid affinity column; 2) applying an aqueous, buffered solutioncomprising said immunoglobulin in monomeric and in aggregated form to ananion exchange material under conditions whereby said immunoglobulindoes not bind to said anion exchange material, and recovering saidimmunoglobulin in monomeric and in aggregated form from said solutionafter the contact with said anion exchange material; and 3) applying anaqueous, buffered solution comprising said immunoglobulin in monomericand in aggregated form to a cation exchange material under conditionswhereby said immunoglobulin in monomeric form does not bind to saidcation exchange material, and recovering said immunoglobulin inmonomeric form from said solution after the contact with said cationexchange material, wherein: said aggregated form is an immunoglobulinmolecule associated either covalently or non-covalently with a least oneadditional immunoglobulin molecule, said anion exchange material is achromatography material comprising only anionic charged groups aschromatographically active substituents, and said cation exchangematerial is a chromatography material comprising only cationic chargedgroups as chromatographically active substituents.
 2. The methodaccording to claim 1, characterized in that said step 3) is achromatographic step operated in flow-through mode comprising applyingan aqueous, buffered solution comprising said immunoglobulin inmonomeric and in aggregated form to a cation exchange material underconditions whereby said immunoglobulin in monomeric form does not bindto said cation exchange material, and recovering said immunoglobulin inmonomeric form from said solution after the contact with said cationexchange material.
 3. The method according to claim 2, characterized inthat said step 2) is a chromatographic step operated in flowthrough-mode comprising applying an aqueous, buffered solutioncomprising said immunoglobulin in monomeric and in aggregated form to ananion exchange material under conditions whereby said immunoglobulindoes not bind to said anion exchange material, and recovering saidimmunoglobulin in monomeric and in aggregated form from said solutionafter the contact with said anion exchange material.
 4. The methodaccording to claim 3, characterized in that said step 1) is achromatographic step operated in bind-and-elute mode comprising applyingan aqueous, buffered solution comprising said immunoglobulin inmonomeric and in aggregated form to an affinity column under conditionswhereby said immunoglobulin binds to said affinity column, andrecovering said immunoglobulin in monomeric and in aggregated form fromsaid affinity column.
 5. The method according to claim 1, characterizedin that in said step 3) the cation exchange material is a membranecation exchange material.
 6. The method according to claim 5,characterized in that said membrane cation exchange material is apolyethersulfone based membrane or a regenerated cellulose basedmembrane modified with sulfonic acid groups or carboxymethyl groups. 7.The method according to claim 1, characterized in that said aqueous,buffered solution of step 3) has a pH value of from pH 5 to pH
 8. 8. Themethod according to claim 1, characterized in that said aqueous,buffered solution of step 3) has a conductivity of from 1.0 to 15.0mS/cm.
 9. The method according to claim 8, characterized in that saidaqueous, buffered solution of step 3) has a conductivity of from 4.0 to10.0 mS/cm.
 10. The method according to claim 1, characterized in thatsaid recovering said immunoglobulin in monomeric form from theflow-through is by a method selected from precipitation, salting out,ultrafiltration, diafiltration, lyophilization, affinity chromatography,or solvent volume reduction to obtain a concentrated solution.
 11. Themethod according to claim 10, characterized in that said recovering isby ultrafiltration, lyophilization, or solvent volume reduction.
 12. Themethod according to claim 1, characterized in that of saidimmunoglobulin obtained from the flow-through of the membrane cationexchange material at least 95% of the immunoglobulin is in monomericfrom.
 13. The method according to claim 1, characterized in that atleast 90% of the immunoglobulin in monomeric form does not bind to thecation exchange material.
 14. The method according to claim 1,characterized in that said aqueous, buffered solution is a solutioncomprising phosphoric acid or salts thereof, citric acid or saltsthereof, or histidine or salts thereof.
 15. The method according toclaim 1, characterized in that said aqueous, buffered solution comprisessodium chloride or potassium chloride.
 16. The method according to claim1, characterized in that the sum of pH value and conductivity in mS/cmof the aqueous, buffered solution in step 3) is in the range of from 9to
 18. 17. The method according to claim 16, characterized in that saidsum is in the range of from 10 to 15.